CN1294192A - Processf or preparing abzyme with activity of thyroxin 5' deiodinase - Google Patents
Processf or preparing abzyme with activity of thyroxin 5' deiodinase Download PDFInfo
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- CN1294192A CN1294192A CN99121250A CN99121250A CN1294192A CN 1294192 A CN1294192 A CN 1294192A CN 99121250 A CN99121250 A CN 99121250A CN 99121250 A CN99121250 A CN 99121250A CN 1294192 A CN1294192 A CN 1294192A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
A process for preparing abzyme with the activity of thyroxin 5' deiodinase includes covalent cross-linking of thyroxin as semi-antigen with ox's serum albumin to obtain full-antigen, preparing antibody with thyroxin binding power by monocloning antibody and chemical modifying to introduce the catalytic center SeH of thyroxin deiodinase to the antigen bindingposition of antibody for giving it with the activity of thyroxin deiodinase. Its advantages are high stability and high activity (290U/mgPr).
Description
The invention belongs to and have the preparation method that thyroxine 5 ' takes off iodine enzyme activity abzyme.
Thyroxine is a kind of necessary hormone of human body, and thyroxine is with numerous disease is relevant clinically.The intravital thyroxine of people exists with two kinds of activity forms: 3,5, and the former propylhomoserin (T of 3 ', 5 '-TA
4) and 3,5, the former propylhomoserin (T of 3 '-Lithyronine
3).T
3Biologic activity be 8 times of T4, be the thyroxinic principal mode of activity in vivo.T4 in all peripheral tissues and the blood is synthetic by Tiroidina, and T3 has the thyroxine 5 ' that comes from peripheral tissues more than 85% to take off the catalysate of iodine enzyme.As seen to take off the iodine enzyme significant for thyroxine performance physiological function for thyroxine 5 '.At present, people find three kinds of thyroxine deiodinases altogether, wherein, I type thyroxine deiodinase (IT45 ' D) be a kind of membranin that contains selenium, extensively be present in positions such as liver, kidney, Tiroidina, muscle, most of T3 is produced by this kind of enzyme catalysis.Nineteen ninety, there is report to adopt gel electrophoresis from related tissue, to be separated to IT45 ' D.Adopt radio isotope Se75 mark, find that IT45 ' D is a kind of selenoenzyme.Berry in 1991 etc. (Nature, 1991,349:438-440) utilization clonal expression technology has been separated to the complementary DNA of IT45 ' D from the mouse liver in the toad ovocyte, wherein contains the TGA codon seleno-cysteine of encoding.Selenium among the IT45 ' D exists with seleno half wide propylhomoserin form, and is enzymatic necessary amino acid.Abzyme is the new research field that grows up in the later stage eighties.It has given the attribute of enzyme with the variable region of immunoglobulin (Ig), is catalytic antibody albumen.1986, on the basis of monoclonal antibody technique, Schultz and Lerner just reported the first monoclonal antibody of hydrolytic enzyme activities that has respectively.In the short more than ten years, the catalytic reaction of abzyme has reached kind more than 70.Hydrophobic pocket modification method is the up-to-date thought of abzyme design.Chinese patent 96112628.0 discloses people such as Ding Lan and has utilized this method successfully to prepare the abzyme with GSH-Px vigor than the high octuple of natural enzyme vigor first, adopting the gsh derivative is haptens, be covalently bonded on the bovine serum albumin with glutaraldehyde, the antibody that has strong bonding force with antigen that obtains that there is hydrophobic pocket in antigen-binding site, with the method for catalytic center by chemomorphosis, introduce antigen-binding site, make antibody have the GSH-Px activity.
The purpose of this invention is to provide a kind of preparation method that thyroxine 5 ' takes off iodine enzyme activity abzyme that has.This method is haptens with thyroxine, is connected with bovine serum albumin, and immune mouse, preparation has the antibody with the thyroxine binding ability, by chemically modified, IT45 ' D catalytic center is introduced antibody, obtains to have the abzyme of IT45 ' D vigor.
Natural thyroxine side chain is two halogenated phenyl ring, has the ability that induces hydrophobic combining environmental at the antigen-binding site of antibody, itself is exactly fine hapten molecule.Because natural thyroxine does not possess immunogenicity, can not stimulate animal to produce antibody, so at first thyroxine is connected on the bovine serum albumin by glutaraldehyde, select individual less, immune mechanism is comparatively clear, and immunologic process is simple relatively, the pure lines Bab/C mouse of easy handling is immune object, by to mouse immune, the immune stimulatory cell makes its secretion have the antibody of thyroxine binding ability.For making the quick division of immunocyte, so that mass preparation antibody, mouse immune cell and myeloma cell are merged the preparation hybridoma carry out cloning, obtain secreting the single cell clone of purpose antibody.Cell strain enlarged culturing with obtaining is injected into mouse peritoneal, and preparation contains the ascites of purpose antibody from mouse peritoneal.By the affinity chromatography separation and purification, purification purpose antibody.Structure according to the catalytic center of thyroxine deiodinase, with the antibody that obtains with TBC by chemically modified, with the hydroxyl activation of phenylmethylsulfonyl fluoride with the Serine of antibody thyroxine combining site, react with sodium hydrogen selenide, catalytic center one SeH of IT45 ' D is introduced the thyroxine combining site of antibody, give antibody I T45 ' vigor of D.Preparation concrete steps of the present invention are as follows:
A. the preparation of holoantigen
With T4 and bovine serum albumin with 30-50: the ratio of 1 (mol ratio) is dissolved in the Tris-HCl damping fluid of pH8-11, and wherein, T4 concentration is that 1-5mg/ml stirs, drip glutaraldehyde gradually, reacted 10-24 hour, add the glycine termination reaction, desalination, freeze-drying.
B. MONOCLONAL ANTIBODIES SPECIFIC FOR
A. immune mouse
Antigen 1-3mg/ml is mixed with the Fu Shi Freund's complete adjuvant at 1: 1, shake up, every Bab/c injected in mice 100-300 μ l carries out second immunisation after two weeks, the 4th to ten week every two weeks with the freund 's incomplete adjuvant immunity, detect antibody with ELISA.
B. merge and Antibody Preparation
Draw the neck method to put to death mouse, get spleen, remove fatty tissue under the aseptic condition in the plate of placing nutrient solution in advance, cell suspension is drawn in crushing, and is centrifugal, abandons supernatant.Use the complete culture solution repetitive scrubbing, obtain 0.5-1.5 * 10
8Individual splenocyte.The myeloma cell who adds 0.1-0.5 multiple amount mixes, and is centrifugal.The nutrient solution of supernatant discarded is drawn 1ml polyoxyethylene glycol (PEG)/methyl-sulphoxide (DMSO) and is added in the cell, mixes 1 minute.
The cell suspension repetitive scrubbing that will contain PEG, the substratum that brings Selection In is being cultivated under 37 degree, 5% gas concentration lwevel on the culture plate.Microscopy after one week carries out ELISA to tangible cell colony hole, gets positive hole gradient dilution, and enlarged culturing is carried out first cloning.Cloning is 3-5 time repeatedly, can obtain single cell clone.
With 0.5-1.5 * 10
6Hybridoma inject the Bab/C mouse peritoneal or adopt extracorporeal culture-ing, in culturing bottle, obtain ascites or culture supernatant.
C. separate purification antibody
With ascites or cell in vitro medium centrifugal, get supernatant and adopt affinity chromatography or ion exchange column purification antibody, freeze-drying.
C. the preparation of abzyme
The antibody lyophilized powder 5-10mg that purifies is dissolved in the phosphoric acid buffer of pH6-8 of 1ml deoxygenation, adds the acetonitrile solution of the phenylmethylsulfonyl fluoride of 10-30 μ l 20mg/ml,
Room temperature vibration 0.5-1 hour is that carrier feeds H with nitrogen
2The 1MNaHSe solution of Se gas 10 minutes or adding 50-200 μ l deoxygenation in advance, the 30-40 degree reacted 20-40 hour under nitrogen protection.Reaction solution is centrifugal, freeze-drying behind the desalination.Promptly make the abzyme lyophilized powder.
People thought always in the past that seleno-protein just played the function of removing free radical and superoxide in vivo, and the discovery of IT45 ' D provides new research field for seleno-protein and selenium in the intravital functional study of people again.Natural IT45 ' D makes it separate purification difficult owing to have characteristics such as lipotropy, content are low, has limited its research and application greatly, and up at present, people are to the understanding of its mechanism hypothesis just also.The appearance of manual simulation's enzyme of IT45 ' D, the effect that selenium is played in this enzyme and the hypothesis of mechanism provide important evidence.One of subacute cretinistic symptom clinically is exactly a T4 content height in the body, and T3 content is low, thereby the metabolism of whole machine body is exerted an influence.The patient of minority endemic goiter is after having replenished iodine, and symptom still can not get alleviating, and these all are considered to, and to take off the iodine enzyme relevant with thyroxine 5 '.Natural enzyme not only is difficult to obtain, and poor stability, and its clinical application and research are restricted.Abzyme has good stability, and characteristics such as easy mass preparation are for treating correlative diseases provides a kind of feasible approach.
Embodiment provided by the invention is as follows:
Embodiment 1:
(1) holoantigen is synthetic
T41mg and bovine serum albumin are dissolved in the ratio of 50: 1 (mol ratio) in the damping fluid of Tris-HCl of pH9.0 20mmol/L, stir, the 1ml glutaraldehyde solution of mol ratio such as dropping gradually, reacted 12 hours, add 1ml10mmol/L glycine termination reaction, with 20mmol/L pH 7.4 phosphoric acid buffer dialysed overnight, with ultraviolet spectroscopy haptens connection amount, each bovine serum albumin connects 30 hapten molecules, and freeze-drying is preserved.
(2) MONOCLONAL ANTIBODIES SPECIFIC FOR
A. immunity
With holoantigen 2mg/ml and Fu Shi Freund's complete adjuvant mixed with 1: 1, shake up, every Bab/c injected in mice 200 μ l carry out second immunisation after two weeks, every two weeks with the freund 's incomplete adjuvant immunity once, merge first three day booster immunization.
B. detection of antibodies
The every hole of enzyme plate is added 100 μ l5% glutaraldehyde solutions, 37 degree insulation 15min, washing pats dry.The T4 solution that adds 100 μ l 1mg/ml is incubated 1 hour.Every hole adds the bovine serum albumin solution of the 0.1mg/ml of 200 μ l, is incubated 1 hour.Add test serum or ascites, be incubated 1 hour.Every hole adds the ELIAS secondary antibody of a unit of activity, is incubated 1 hour.Add substrate reactions liquid 100 μ l, 37 degree react half an hour, and every hole adds 50 μ l 2mmol/L sulphuric acid soln termination reactions, detects with microplate reader.
C. the preparation of fusion and antibody
The mouse of serum test positive with drawing the neck method to put to death, is got spleen, remove fatty tissue under the aseptic condition in the plate of placing nutrient solution in advance, cell suspension is drawn in crushing, and is centrifugal, abandons supernatant.Use the complete culture solution repetitive scrubbing, obtain 1.2 * 10
8Individual splenocyte.The myeloma cell who adds 0.1 multiple amount mixes, and is centrifugal.The nutrient solution of supernatant discarded is drawn 1ml PEG/DMSO and is added in the cell, mixes 1 minute.
The cell suspension repetitive scrubbing that will contain PEG, the substratum that brings Selection In is being cultivated under 37 degree, 5% gas concentration lwevel on the culture plate.Microscopy after one week carries out ELISA to tangible cell colony hole, gets positive hole gradient dilution, and enlarged culturing is carried out first cloning.Cloning is 3 times repeatedly, obtains single cell clone.
With 1 * 10
6Hybridoma inject the Bab/C mouse peritoneal, obtain ascites.
D. the separation of antibody is purified
With centrifugal 15 minutes of the ascites 6000rpm/min in the mouse peritoneal, get supernatant, use 60% ammonium sulphate precipitation, centrifugal 20 minutes of 6000rpm/min abandons supernatant, will precipitation with the PBS dissolving of pH7.0, cross G-25 post desalination.Elutriant is separated purification through γ-Protein A affinity column, and freeze-drying obtains refining antibody.
(3) preparation of abzyme and vitality test
A. the preparation of abzyme
The antibody lyophilized powder 5mg that purifies is dissolved among the PBS of pH7.0 of 1ml deoxygenation; the acetonitrile solution that adds the phenylmethylsulfonyl fluoride of 20 μ l 20mg/ml; room temperature was vibrated 0.5 hour, added the 1MNaHSe solution of 100 μ l deoxygenations in advance, and 30 degree reacted 40 hours under nitrogen protection.8000rpm/min is centrifugal with reaction solution, freeze-drying behind the G-25 desalination.Make the abzyme lyophilized powder.
B. abzyme vitality test
Adopting radioimmunoassay (RIA) method to survey lives
Abzyme is dissolved among the 700 μ lPBS of pH 7.4, the DTT that adds 100 μ l 10mM, add 10 μ M T4100 μ l, the BSA solution 100 μ l that add 1f/100ml, 37 degree react half an hour, add the long-pending pre-cooled ethanol of diploid, the centrifugal 10min of 6000g, get supernatant, reaction solution is diluted to 1-30/100ml T4, the adding of reaction diluent is coated with in the test tube of T4 or T3 in advance, temperature was bathed 0.5 hour, add T4 or the T3 solution that has radioactivity I125 mark again, temperature was bathed 0.5-1 hour, measured the radiation dose of every pipe.With the T4 and the T3 production standard curve of different concns, in typical curve, find out corresponding T4, T3 concentration under the same terms.
Adopting radioimmunology to record the abzyme vigor is 275U/mgPr (the pmol number of U-per minute conversion of substrate)
Embodiment 2:
(1) holoantigen is synthetic
With T4 and bovine serum albumin with the mixed of 30: 1 (mol ratio) in the Tris-HCl of pH10 20mmol/L damping fluid, stir, drip glutaraldehyde gradually, reacted 15 hours, other reaction conditions is with (1) among the embodiment 1.
(2) MONOCLONAL ANTIBODIES SPECIFIC FOR
A. with holoantigen 3mg/ml and the Fu Shi Freund's complete adjuvant mixed with 1: 1, shake up, every Bab/c injected in mice 200 μ l carry out second immunisation after two weeks, every two weeks with the freund 's incomplete adjuvant immunity once, merge first three day booster immunization.
B. detection of antibodies
With (2) b among the embodiment 1.
C. the preparation of fusion and antibody
The mouse of serum test positive with drawing the neck method to put to death, is got spleen, remove fatty tissue under the aseptic condition in the plate of placing nutrient solution in advance, cell suspension is drawn in crushing, and is centrifugal, abandons supernatant.Use the complete culture solution repetitive scrubbing, obtain 1.5 * 10
8Individual splenocyte.The myeloma cell who adds 0.3 multiple amount mixes, and is centrifugal.The nutrient solution of supernatant discarded is drawn 1mlPEG/DMSO and is added in the cell, mixes 1 minute.
The cell suspension repetitive scrubbing that will contain PEG, the substratum that brings Selection In is being cultivated under 37 degree, 5% gas concentration lwevel on the culture plate.Microscopy after one week carries out ELISA to tangible cell colony hole, gets positive hole gradient dilution, and enlarged culturing is carried out first cloning.Cloning is 4 times repeatedly, obtains single cell clone.
With 1.5 * 10
6Hybridoma inject the Bab/C mouse peritoneal, obtain ascites.
D. the separation of antibody is purified
With (2) d among the embodiment 1.
(3) preparation of abzyme and vitality test
A. the preparation of abzyme
The antibody lyophilized powder that 10mg is purified is dissolved among the PBS of pH8 of 1ml deoxygenation, adds the acetonitrile solution of the phenylmethylsulfonyl fluoride of 30 μ l20mg/ml, room temperature vibration 50 minutes.The 1mol/L NaSeH that adds 200 μ l deoxygenations in advance, 40 degree reaction 30 hours under nitrogen protection, with 8000 rpms of reaction solutions centrifugal after, through G-25 post desalination, freeze-drying makes the abzyme lyophilized powder.
B. abzyme vitality test
Adopting radioimmunology to record the abzyme vigor is 290U/mgPr
Embodiment 3
(1) preparation of holoantigen
With T4 and bovine serum albumin with the mixed of 40: 1 (mol ratio) in the Tris-HCl of pH11 20mmol/L damping fluid, drip glutaraldehyde gradually, stirred 24 hours, other reaction conditions is with (1) among the embodiment 1.
(2) MONOCLONAL ANTIBODIES SPECIFIC FOR
A. with holoantigen (1mg/ml) and the Fu Shi Freund's complete adjuvant mixed with 1: 1, shake up, every Bab/c injected in mice 300 μ l carry out second immunisation after two weeks, every two weeks with the freund 's incomplete adjuvant immunity once, merge first three day booster immunization.Be total to immune six weeks.
B. detection of antibodies
With (2) b among the embodiment 1.
C. the preparation of fusion and antibody
The mouse of serum test positive with drawing the neck method to put to death, is got spleen, remove fatty tissue under the aseptic condition in the plate of placing nutrient solution in advance, cell suspension is drawn in crushing, and is centrifugal, abandons supernatant.Use the complete culture solution repetitive scrubbing, obtain 0.5 * 10
8Individual splenocyte.The myeloma cell who adds 0.5 multiple amount mixes, and is centrifugal.The nutrient solution of supernatant discarded is drawn 1ml PEG/DMSO and is added in the cell, mixes 1 minute.
The cell suspension repetitive scrubbing that will contain PEG, the substratum that brings Selection In is being cultivated under 37 degree, 5% gas concentration lwevel on the culture plate.Microscopy after one week carries out ELISA to tangible cell colony hole, gets positive hole gradient dilution, and enlarged culturing is carried out first cloning.Cloning is 3 times repeatedly, obtains single cell clone.
With 0.5 * 10
6Hybridoma inject the Bab/C mouse peritoneal, obtain ascites.
D. the separation of antibody is purified
With (2) d among the embodiment 1.
(3) preparation of abzyme and vitality test
A. the preparation of abzyme
The antibody lyophilized powder that 5mg is purified is dissolved among the PBS of pH6 of 1ml deoxygenation, adds the acetonitrile solution of the phenylmethylsulfonyl fluoride of 10 μ l 20mg/ml, room temperature vibration 1 hour.The 1mol/L NaSeH that adds 50 μ l deoxygenations in advance, 40 degree reaction 20 hours under nitrogen protection, with 8000 rpms of reaction solutions centrifugal after, through G-25 post desalination, freeze-drying makes the abzyme lyophilized powder.
B. abzyme vitality test
Adopting radioimmunology to record the abzyme vigor is 230U/mgPr
Claims (1)
1. one kind has the preparation method that thyroxine 5 ' takes off iodine enzyme activity abzyme, it is characterized in that adopting following steps to finish:
A. the preparation of holoantigen
With T4 and bovine serum albumin with 30-50: the ratio of 1 (mol ratio) is dissolved in the Tris-HCl damping fluid of pH8-11, and wherein, T4 concentration is that 1-5mg/ml stirs, drip glutaraldehyde gradually, reacted 10-24 hour, add the glycine termination reaction, desalination, freeze-drying;
B. MONOCLONAL ANTIBODIES SPECIFIC FOR
A. immune mouse
Antigen 1-3mg/ml is mixed with the Fu Shi Freund's complete adjuvant at 1: 1, shake up, every Bab/c injected in mice 100-300 μ l carries out second immunisation after two weeks, the 4th to ten week every two weeks with the freund 's incomplete adjuvant immunity, detect antibody with ELISA;
B. merge and Antibody Preparation
Draw the neck method to put to death mouse, get spleen, remove fatty tissue under the aseptic condition in the plate of placing nutrient solution in advance, cell suspension is drawn in crushing, and is centrifugal, abandons supernatant, uses the complete culture solution repetitive scrubbing, obtains 0.5-1.5 * 10
8Individual splenocyte, the myeloma cell of adding 0.1-0.5 multiple amount mixes, and is centrifugal, and the nutrient solution of supernatant discarded is drawn 1ml polyoxyethylene glycol (PEG)/methyl-sulphoxide (DMSO) and is added in the cell, mixes 1 minute;
The cell suspension repetitive scrubbing that will contain PEG, substratum brings Selection In, cultivating under 37 degree, 5% gas concentration lwevel on the culture plate, microscopy after the week carries out ELISA to tangible cell colony hole, get positive hole gradient dilution, enlarged culturing is carried out first cloning, and cloning is 3-5 time repeatedly, obtain single cell clone, with 0.5-1.5 * 10
6Hybridoma inject the Bab/C mouse peritoneal or adopt extracorporeal culture-ing, in culturing bottle, obtain ascites or culture supernatant;
C. separate purification antibody
With ascites or cell in vitro medium centrifugal, get supernatant and adopt affinity chromatography or ion exchange column purification antibody, freeze-drying;
C. the preparation of abzyme
The antibody lyophilized powder 5-10mg that purifies is dissolved in the phosphoric acid buffer of pH6-8 of 1ml deoxygenation, adds the acetonitrile solution of the phenylmethylsulfonyl fluoride of 10-30 μ l 20mg/ml,
Room temperature vibration 0.5-1 hour, the 1MNaHSe solution of adding 50-200 μ l deoxygenation in advance, the 30-40 degree, reaction is 20-40 hour under nitrogen protection, and reaction solution is centrifugal, and freeze-drying behind the desalination makes the abzyme lyophilized powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99121250A CN1294192A (en) | 1999-10-22 | 1999-10-22 | Processf or preparing abzyme with activity of thyroxin 5' deiodinase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99121250A CN1294192A (en) | 1999-10-22 | 1999-10-22 | Processf or preparing abzyme with activity of thyroxin 5' deiodinase |
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| CN1294192A true CN1294192A (en) | 2001-05-09 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102372775A (en) * | 2010-08-20 | 2012-03-14 | 富士胶片株式会社 | Method for producing conjugate of thyroxine and albumin |
-
1999
- 1999-10-22 CN CN99121250A patent/CN1294192A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102372775A (en) * | 2010-08-20 | 2012-03-14 | 富士胶片株式会社 | Method for producing conjugate of thyroxine and albumin |
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